Probing binding mechanism of interleukin-6 and olokizumab: in silico design of potential lead antibodies for autoimmune and inflammatory diseases

Computer-aided antibody engineering has been successful in the design of new biologics for disease diagnosis and therapeutic interventions. Interleukin-6 (IL-6), a well-recognized drug target for various autoimmune and inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis, was investigated in silico to design potential lead antibodies. Here, crystal structure of IL-6 along with monoclonal antibody olokizumab was explored to predict antigen–antibody (Ag???Ab)-interacting residues using DiscoTope, Paratome, and PyMOL. Tyr56, Tyr103 in heavy chain and Gly30, Ile31 in light chain of olokizumab were mutated with residues Ser, Thr, Tyr, Trp, and Phe. A set of 899 mutant macromolecules were designed, and binding affinity of these macromolecules to IL-6 was evaluated through Ag???Ab docking (ZDOCK, ClusPro, and Rosetta server), binding free-energy calculations using Molecular Mechanics/Poisson Boltzman Surface Area (MM/PBSA) method, and interaction energy estimation. In comparison to olokizumab, eight newly designed theoretical antibodies demonstrated better result in all assessments. Therefore, these newly designed macromolecules were proposed as potential lead antibodies to serve as a therapeutics option for IL-6-mediated diseases.     

The determination of spatial pattern inDictyostelium discoideum

Free-living amoebae of the cellular slime mouldDictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case ofDictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) ‘coin tossing’, whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) ‘chemical kinetics’, whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other; and (iii) ‘positional information’, in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arisesin situ     

Purification and characterization of ATP:AMP phosphotransferase from Mycobacterium marinum

ATP:AMP phosphotransferase (EC 2.7.4.3) (adenylate kinase) has been purified 1746-fold from Mycobacterium marinum (ATCC 927) by successive column chromatography on DEAE-cellulose (DE-53), Reactive Blue agarose, Sephadex G-75, hydroxyapatite and, finally, DEAE-Sephadex A-50. The final enzyme preparation had a specific activity of 576 mumol/min per mg protein with an overall yield of 51%. The preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was estimated to have an Mr of 29500 and an isoelectric point of 6.7, properties which generally resemble those of the mitochondrial enzyme. Indeed, the two enzymes failed to separate when subjected to polyacrylamide gel electrophoresis under denaturing conditions. The extinction coefficient (at 276 nm) was calculated to be 3.114 X 10(4) M-1 X cm-1 and E1%1cm = 10.556. Adenylate kinase was present at a concentration of 0.06 mg/g (wet weight) bacteria. Enzyme was stable for months in 60% glycerol in the freezer; at 4 degrees C, less than 5% of the activity was lost over a 7 day period.     

Kinetic aspects of the interaction of horse heart cytochrome c with sodium dodecyl sulfate

The sodium dodecyl sulfate (SDS) concentration dependence of spectral changes in circular dichroism (CD) and in absorbance of cytochrome c were examined in the far-ultraviolet region, aromatic region, and the Soret band. The Soret peak obtained in 0.60 mM SDS was nine times greater than that of the native state. (The critical micelle concentration, CMC, of SDS was 2.2 mM in the phosphate buffer used.) The results indicated that the drastic change at the Soret band did not accompany the corresponding large-scale change in secondary structure of the protein. In the stopped-flow measurements, two and three processes were followed at 406 nm below and above the CMC, respectively. At 289 nm only one process was observed, and this corresponded to the second process at 406 nm. Therefore, the second process at 406 nm was considered to be a change in tertiary structure around the heme group. The first process and the third process seemed to reflect a change in the heme environment; the former appeared to be due to a solvent effect and the latter due to a binding effect of a large number of dodecyl sulfate ions.